Constitutive Turbodomains enhance expansion and antitumor activity of allogeneic BCMA CAR T cells in preclinical models

The magnitude of CAR T cell expansion has been associated with clinical efficacy. Although cytokines can augment CAR T cell proliferation, systemically administered cytokines can result in toxicities. To gain the benefits of cytokine signaling while mitigating toxicities, we designed constitutively active synthetic cytokine receptor chimeras (constitutive Turbodomains) that signal in a CAR T cell–specific manner. The modular design of Turbodomains enables diverse cytokine signaling outputs from a single homodimeric receptor chimera and allows multiplexing of different cytokine signals. Turbodomains containing an IL-2/15Rβ–derived signaling domain closely mimicked IL-15 signaling and enhanced CAR T cell potency. Allogeneic TurboCAR T cells targeting BCMA showed no evidence of aberrant proliferation yet displayed enhanced expansion and antitumor activity, prolonging survival and preventing extramedullary relapses in mouse models. These results illustrate the potential of constitutive Turbodomains to achieve selective potentiation of CAR T cells and demonstrate the safety and efficacy of allogeneic BCMA TurboCAR T cells, supporting clinical evaluation in multiple myeloma.

FACS sorting, BCMA CAR T cells were stained using the Zombie NIR Fixable Viability Kit (BioLegend, San Diego, CA), followed by the anti-idiotype-PE. Viable CAR + cells were bulk sorted on the FACS Aria II Cell Sorter (BD Biosciences, San Jose, CA).

Lentivirus production, concentration, titration and quantification
To generate lentiviral supernatants, HEK293T cells were plated at 0.45 million cells per ml in 2 ml of DMEM (Gibco, Thermo Fisher Scientific, Waltham, MA) supplemented with 10% FBS (GE Healthcare, Pittsburgh, PA) per well of a 6-well plate on Day -1. On Day 0, the lentivirus was prepared by mixing lentiviral packaging vectors 1.5 μg psPAX2, 0.5 μg pMD2G, and 0.5 μg of the appropriate transfer CAR/TurboCAR vector in 250 μl Opti-MEM per well of the 6-well plate ("DNA mix"). 10 μl Lipofectamine 2000 in 250 μl Opti-MEM was incubated at room temperature for 5 minutes and then added to the DNA mix. The mixture was incubated at room temperature for 20 minutes and the total volume of 500 μl was slowly added to the sides of the wells containing HEK293T cells. On Day 1, Lipofectamine-containing supernatants were removed and replaced with X-Vivo15 medium (Lonza, Basel, Switzerland), supplemented with 10% FBS (GE Healthcare, Pittsburgh, PA). On Day 2, crude lentiviral supernatants were harvested, filtered through a 0.45 μm filter, and were either used fresh for human CAR T cell generation, or concentrated 25x using Lenti-X Concentrator (Takara Bio, Shiga, Japan) according to the manufacturer's instructions and stored at -80°C until use. Where applicable, lentiviral titration was performed on Jurkat cells. Briefly, on Day 0, 2×10 4 Jurkat cells were transduced with serially diluted lentivirus in 96-well U-bottom plates. On Day 2, the percentage of CAR + Jurkat cells was determined by flow cytometry. Titer was calculated using the volume of lentivirus yielding 10% CAR + cells.

Complement-dependent cytotoxicity (CDC) assay
1x10 5 cells were incubated in RPMI 1640 medium supplemented with 10% FCS for 1 h in the presence of 12.5% baby rabbit complement (Bio-Rad, Hercules, CA) and rituximab at 0, 10, 30, and 100 μg/ml. Cells were then collected by centrifugation, washed with FACS buffer, and stained for flow cytometry analysis.
The percentages of depleted CAR + cells were normalized between 0 and 100% relative to wells without antibody (set to 0% depletion). CDC was expressed as the increase in the % of depleted CAR + cells relative to the control using the formula 100 -[100 x (CAR + cells test /CAR + cells control)].

Antibody-dependent cellular cytotoxicity (ADCC) assay
Natural killer (NK) cells were isolated using StemCell Technologies NK Cell Enrichment Cocktail following the manufacturer's protocol. NK cells were then counted, suspended at 4x10 6 cells/ml in RPMI 1640 medium supplemented with 10% FCS, and activated using 1000 IU/ml IL-2 for 2 days in a humidified incubator at 37°C and 5% CO2. On Day 2, NK cells were collected by centrifugation and suspended in the same medium. CAR T cells were labeled with CellTrace Violet dye following the manufacturer's protocol and co-cultured with NK cells at an E:T of 10 for one hour. Rituximab antibody was added at increasing concentrations ranging from 0.1 to 100 μg/ml. Cells were then collected by centrifugation, washed with FACS buffer, and stained for flow cytometry analysis. The percentages of depleted CAR + cells were normalized between 0 and 100% relative to wells with CAR T cells without antibody and NK cells (set to 0% depletion). ADCC was expressed as the increase in the % of depleted CAR + cells relative to the control using the formula 100 -[100 x (CAR + cells test /CAR + cells control)]. Two NK cell donors and CAR T cells produced from 3 donors were tested in this study.   increase in live target cells was determined by normalizing target cell counts at each timepoint to that at time=0. Data are mean ± SEM of triplicate wells from one representative of two donors. Statistical significance determined by ordinary one-way ANOVA with Dunnett's test. *P < 0.05, ****P < 0.0001, ns: not significant.     cells/mouse) and tumor volume was measured using calipers. When tumors reached approximately 180 mm 3 , a mix of CBR-labeled and unlabeled TurboCAR T cells (0.5x10 6 CBR + CAR + cells and 9.5x10 6 unlabeled CAR + cells) was injected intravenously into mice and bioluminescence was measured for several weeks. Images were taken at the indicated timepoints and are representative of the group.

Fig. S9: Allogeneic BCMA TurboCAR T cells can be depleted by rituximab via ADCC and CDC. (A)
BCMA TurboCAR T cells were incubated with activated NK cells and various concentrations of the anti-CD20 antibody rituximab or an isotype control antibody (100 μg/ml). After one hour, cells were stained for CAR expression and analyzed by flow cytometry to determine the percentages of depleted CAR + cells relative to wells containing CAR T cells cultured without antibody and NK cells. The graph shows the percentage of depleted CAR + cells (mean ± SEM; n=3 donors). Statistical analysis using-2-way ANOVA and Sidak's multiple comparison revealed no significant difference between BCMA TurboCAR T cells and BCMA CAR T cells. NK cells from two donors were tested in the assay, and data from one representative donor are shown. (B) BCMA TurboCAR T cells were incubated with baby rabbit complement and various concentrations of rituximab or an isotype control antibody (100 μg/ml) for one hour. Cells were washed and stained for CAR expression for flow cytometry analysis. The graph shows the percentage of depleted CAR + cells (mean ± SEM; n=3 donors). No significant differences in CDC were observed between BCMA